Novel method for purification of recombinant proteins

ABSTRACT

Purification of poly-amino acid-tagged recombinant proteins has been improved by the use of a carboxymethylated aspartate ligand complexed with a third-block transition metal having an oxidation state of 2 + and a coordination number of 6. A method for synthesizing the metal ion-CM-Asp complex is also described. Further, the metal ion-CM- Asp complex can be used for screening protein finction.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of co-pendingapplication Ser. No. 08/698,747, filed Aug. 16, 1996.

BACKGROUND OF THE INVENTION

[0002] Immobilized metal ion affinity chromatography (IMAC) was fsssintroduced by Porath (orath, J., J. Carlsson, I. Olsson, G. Beifrage[1975] Nature 258:598-599.) under the term metal chelate chromatographyand has been previously reviewed in several articles (Porath, J. [1992]Protein Putification and Expression 3:263-281; and articles citedtherein). The IMAC purification process is based on the employment of achelating matrix loaded with soft metal ions such as Cu²⁺ and Ni²⁺.Electron-donating groups on the surface of proteins, especially theimidazole side chain of histidine, can bind to the non-coordinated sitesof the loaded metal. The interaction between the electron donor groupwith the metal can be made reversible by lowering the pH or bydisplacement with inidazole. Thus, a protein possessingelectron-donating groups such as histidine can be purified by reversiblemetal complex/protein interactions.

[0003] Several different metal chelating ligands have been employed inIMAC to purify proteins. Iminodiacetic acid (IDA) ligand is a tridentateand thus anchors the metal with only three coordination sites (Porath,J., B. Olin [1983] Biochemistzy 22:1621-1630). Because of the weakanchoring of the metal, metal leakage has been known to occur. Thetris(carboxymethyl)ethylenediamine (TED) ligand is pentadentate andforms a very strong metal-chelator complex. The disadvantage of this isthat proteins are bound very weakly since only one valence is left forprotein interaction. Nitrilo triacetic acid (NTA) is a tetradentateligand which attempts to balance the metal anchoring strength withmetal-ion protein interaction properties (Hochuli, E., H. Dobeli, A.Schacher [1987] J Chromatography 411:177-184). Other chelating ligandshave been reported and are mentioned. See, e.g., Porath (1992), supra.However, these ligands also have certain disadvantages, includingdecreased bonding capacity, decreased specificity, and increased metalleakage.

[0004] In 1991, Ford et al. (Ford, C., I. Suomninen, C. Glatz [1991]Protein Expression and Purification 2:95-107) described proteinpurification using IMAC technology (Ni-NTA ligand) as applied torecombinant proteins having tails with histidine residues (polyhistidinerecombinant proteins). This method takes advantage of the fact that twoor more histidine residues can cooperate to form very strong metal ioncomplexes. The NTA chelating ligand inimobilized on agarose and loadedwith Ni²⁺ has been useful in this method (Hochuli et al., supra; U.S.Pat. No. 5,047,513). It is available commercially through Qiagen, Inc.(Chatsworth, CA). However, this resin has the disadvantage that theinterchanges between metal ions and poly-histidine recombinant proteinsare not optimal. Metal leakage can occur, and background proteins cansometimes contaminate purification of recombinant proteins.

[0005] A metal chelating gel, i.e., carboxymethylated aspartate (CM-Asp)aarose complexed with calcium, has been used for purifying nativecalcium-binding proteins (Mantovaara, T., H. Pertoft, J. Porath [1989]Biotechnology and Applied Biochemistry 11:564-570; Mantovaara, T., H.Pertoft, J. Porath [1991] Biotechnology and Applied Biochemistry13:315-322; Mantovaara, T., H. Pertoft, J. Porath [1991] Biotechnologyand Applied Biochemistry 13:120-126). However, the Ca²⁺-CM-Asp complexdescribed by Mantovaara et al. has among its disadvantages that it doesnot bind strongly to histidine-tagged recombinant proteins. Anotherdisadvantage, in addition to this inferior binding property, is itsnon-selectivity for histidine tags.

[0006] By contrast, the subject invention comprises the CM-Asp chelatingligand complexed to a transition metal in an octahedral geometry(coordination number of 6). In this unique configuration, the metalcomplex can be advantageously suited for purification of poly-histidinefused recombinant proteins. This is a novel use of the CM-Asp ligand andis part of the subject of the invention herein described.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention concerns a novel IMAC purification methodwhich employs immobilized carboxymethylated aspartate (CM-Asp) ligandsspecifically designed for purification of recombinant proteins fusedwith poly-histidine tags. The new purification method is based upon theCM-Asp chelating matrix having the following structure:

[0008] A general description of the matrix used in the invention andillustrated above is:

[0009] When R4-Pl-R=-IH:

[0010] M=transition metal ion in a 2+oxidation state with a coordinationnumber of 6;

[0011] R₁=a linking arm connecting the nitrogen atom of CM-Asp with R₂;

[0012] R₂=a functional linking group through which CM-Asp llnkng arm R₁is connected to R₃;

[0013] R₃=a polymer matrix, e.g., those polymer matrices typically usedin affinity or gel chromatography.

[0014] When RI-R₂-R₃=H:

[0015] R4=a linking arm connecting the methylene carbon atom of thecarboxymethyl group of CM-Asp with R₅;

[0016] R5=a functional linldng group through which CM-Asp linking arm R4is connected to R6;

[0017] R6=a polymer matrix, e.g., those polymer matrices typically usedin affinity or gel chromatography. In a preferred embodiment:

M=Fe ²⁺, Co ²⁺ , Ni ²⁺ , Cu ²⁺ or Zn ²⁺;

[0018] R₁=CH₂CH(OH)CH₂-, CH,(OH)CH₂-O-CH₂CH(OH)CH₂-;

[0019] -CH₂)₄NHCH₂CH(OH)CH-, and {CH,)₂NHCH₂CH(OH)CH₂-;

[0020] R₂ =O, S, or NH; and

[0021] R₃ =agarose.

[0022] In a particularly preferred embodiment:

[0023] M=Co²+;

[0024] R₁=CH₂CH(OH)CH₂;

[0025] R₂ =O; and

[0026] R3 =agarose, cross-linked.

[0027] Prior to loading the 6XMis recombinant protein to the resin,recombinant cells can be lysed and sonicated. The lysate can then beequilibrated with an aqueous buffer (pH 8) which itself does not formchelates with the metal. An example of an aqueous that can be used atthis step in the described procedure is 50 mM sodium phosphate (pH8.0)/10 nuM Tris-HCI (pH 8.0)/100 mM NaCl, or the like. Theequilibration buffer can contain denaturing agents or detergents, e.g.,10% “TRITON X-100,” 6 M guanidinium HCI, or the like. After binding theprepared 6XHis recombinant protein on the metal CM-Asp chelating resin(the “CM-Asp resin complex), the protein-bound resin is washed at pH 7.0or 8.0. The elution of the protein can be carried out at a constant pHor with a descending pH gradient. In a preferred embodiment, proteinelution can be achieved at a pH of about 6.0 to about 6.3. Altematively,the 6XHis recombinant protein bound to the CM-Asp chelating resin can bewashed with low concentrations (less than 100 MM) of imidazole at pH 8.0and then eluted by increasing the imidazole concentration to 40-100 mM.

[0028] Also included as an aspect of the subject invention is ascaled-up synthesis of the CM-Asp derivatized agarose chelating resin.It is an improved version of a previously reported small scalepreparation (Mantovaara, T., H. Pertoft, J. Porath [1991] BiotechnologyandAppliedBiochemist?y 13:315-322). The improvement includes particularconditions for oxirane-agarose formation, temperature controlledconjugation of aspartic acid to the oxirane-agarose, and high ionicstrength washing to remove extraneously bound metals. These conditions,temperatures, and ionic concentrations are described in detail herein.

[0029] An additional application of the subject invention includesscreening for protein function on a microtiter plate or filter. Theadditional applications for the subject invention also includeprotein-protein interaction studies, as well as antibody and antigenpurification. For example, by immobilization of the Co²⁺ moiety onto96-well plates by CM-Asp, such plates can be used for quantitation of6Xistidine-tagged protein, protein-protein interaction studies,diagnostic screening for diseases, antibody screening, antagonist andagonist screening for drugs, and reporter gene assays. Co²⁺ can also beimmobilized onto a membrane, e.g., a nylon membrane, by CM-Asp, whichcan be used to lift proteins from expression libraries to make proteinlibraries from cells. The membranes also can be used for screening ofengineered enzymes. Application of the subject invention can also beextended to purification of any interacting molecule, e.g., nucleicacids or small co-factors.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an outline illustrating aprocess for purifying recombinant 6XHis protein using CM-Asp chelatingresin.

[0030] FIGS. 2A-2B show a comparison of Co²+CM-Asp chelating resin withNi-NTA on 6XHis prepro-a-factor purification under denaturing conditionsusing pH gradient.

[0031] Legend for FIGS. 2A-2B: lane 1: crude lysate; lane 2:flowthrough; lane 3: washed with 6 M Gu-HCI, 0.1 M NaH₂PO₄, pH 8.0; lane4: washed with 8 M urea, 0.1 M NaH₂PO₄; lane 5: washed with 8 M urea,0.1 M NaH₂PO₄, pH 8.0; lane 6: deluted with 8 M urea, 0.1 M NaH₂PO₄, pH6.3; lane 7: deluted with 8 M urea, 0.1 M NaH₂PO₄, pH 6.3; lane 8:deluted with 8 M urea, 0.1 M NaH₂PO₄, pH 6.3; lane 9: deluted with 8 Murea, 0.1 M NaH₂PO₄, pH 5.9; lane 10: deluted with 8 M urea, 0.1 MNaH₂PO₄, pH 4.5; lane 11: deluted with 6 M Gu-HCI, 0.1 M NaH₂PO₄, 0.2 Macetic acid; lane M: MW size markers.

[0032]FIG. 3 shows 6XHis tagged DHFR purification by Co²+CM-Aspchelating resin under native conditions. Legend: Lane 1: clarifiedlysate; lane 2: flowdtrough; lane 3: first wash; lane 4: third wash;lane 5: DHFR final elution.

[0033]FIG. 4 shows 6XHis tagged DHFR purification by Co²⁺ CM-Aspchelating resin under denaturing conditions. Legend: Lane 1: clarifiedlysate; lane 2: flowthrough; lane 3: first pH 7.0 wash; lane 4: secondpH 7.0 wash; lane 5: D}IR, first pH 6.0 elution; lane 6: DEFR, second pH6.0 elution.

[0034]FIG. 5 shows 6XHis tagged DEER purification by Co²⁺ CM-Aspchelating resin under native conditions with increasing concentrationsof P-mercaptoethanol. Legend: lane 1: 20 gl of cell lysate; lanes 2, 4,6, and 8: 20 ll of flowthrough; lanes 3, 5, 7, and 9: 5 [lI of eluant.

[0035]FIG. 6 shows yields of 6XHis DHFR from cell lysates purified byCo²⁺ CM-Asp chelating resin versus Ni-NTA in the presence ofP-mercaptoethanol. Protein concentrations were deternined by Bradfordassay. Yields are expressed as a percentage of total protein in the celllysate.

[0036] FIGS. 7A-7B show purification of 6XNis GFP by Co²⁺ CM-Aspchelating resin under native conditions. The GFP bands were detectedusing Clontechls chemiluminescence Western Exposure Kit and overnightexposure to x-ray film.

[0037] Legend: lane 1: clarified lysate; lane 2: flowthrough; lane 3:first wash; lane 4: first elution; lane 5: second elution; lane 6: thirdelution; lane 7: fourth elution.

[0038]FIG. 8 shows biological activity of 6XFis GFP purified by Co²⁺CM-Asp chelating resin. Leend: tube 1: cell lysate; tube 3: flowthrough;tube 3: wash; tube 4: first elution; tube 5: second elution; tube 6:third elution.

DETAILED DISCLOSURE OF THE INVENTION

[0039] The subject method, which employs a CM-Asp metal chelatingcomplex, can advantageously be used for purification of recombinantproteins having a polyhistidine tail or “tag.”

[0040] According to one embodiment of the subject invention, a resinligand, e.g. CM-Asp, is complexed to a metal other than Ca²⁺, forming aCM-Asp-metal complex. Preferably, the CM-Asp ligand used in the subjectinvention is complexed with one of the transition metals (known as athird-block transition metal), e.g., Fe²⁺, Co²⁺ , Ni²⁺ , Ce, or Zr?+inan octahedral geometry. Other polymer matrices, e.g., polystyrene (as inmicrotiter plates), nylon (as in nylon filters), SEPHAROSE (Pharmacial,Uppeala, Sweden) or the like, can be used with the subject invention andwould readily be recognized by persons of ordinary skill in the art. Thepoly-histidine tag possesses “neighboring” histidine residues which canadvantageously allow the recombinant protein to bind to these transitionmetals in a cooperative manner to form very strong metal ion complexes.This cooperative binding refers to what is commonly known in the art asa “neighboring histidine effect.” For purposes of the subject invention,and as would be understood by a person of ordinary skill in the art, a“strong” or “very strong” metal ion complex refers to the bond strengthbetween the metal ion and the chelating ligand. A strong or very strongmetal ion complex, for example, allows little or essentially no metalleakage from the complex so that the purified protein, e.g., arecombinant protein having a polyhistidine tag, is not contaminated withundesired or “background” protein from a mixture being purified.

[0041] The CM-Asp metal complex offers two available valencies that canform strong and reversible metal complexes with two adjacent histidineresidues on the surface of the recombinant protein. Another advantage tousing the CM-Asp ligand is its ability to strongly anchor the metal ionwhereby metal ion leaking can be virtually eliminated compared to metalleakage observed for other complex binding agents, e.g., Ni-NTA. In amore preferred embodiment, Co²⁺ can be used as the transition metal withCM-Asp. The Co²⁺ -CM-Asp can be less sensitive to reducing agents, suchas P-mercaptoethanol. Metal ion leakage has been shown to remain low,even negligible, in the presence of up to 30 mM P-mercaptoethanol.

[0042] One embodiment of the purification process of the subjectinvention is as follows:

[0043] 1. Prepare lysate/sonicate containing recombinant 6XHis proteinaccording to standard procedures and techniques well known in the art.

[0044] 2. Bind 6XIis protein onto metal-loaded CM-Asp chelating resin atslightly basic pH, e.g., about pH 8.0.

[0045] 3. Wash protein/resin complex at the same basic pH (about pH8.0). Optional washes at a pH of about 7.0 or with imidazole additivecan also be included.

[0046] 4. Elute pure recombinant 6XHis protein with an elution bufferhaving a pH of about 6.0-6.3 or, in the alternative, an elution bufferhaving apH of about 8.0, plus about 40 to about 100 mM imidazole.

[0047] The steps involved in a preferred embodiment of the purificationprocess of the subject invention are illustrated in FIG. 1. The subjectprocess can be employed batchwise, in spin columns, and in large-scalecontinuous-flow columns.

[0048] Buffers used in the above procedures are standard bufferstypically used in similar procedures, with appropriate adjustments andmodifications made as understood in the art. For example, a high ionicstrength buffer, e.g., 50 mM phosphate/ 0 mM Tris/1 00 mM NaCI can beused, with the pH adjusted as needed. The phosphate salt component canrange from a concentration of 10-100 mM; Tris from 5-25 mM; and NaClfrom 50-200 mM.

[0049] Optimal elution conditions depend on the type of impurities, theamount of protein to be purified, and unique properties of the protein,and are determined on a case-by-case basis as would be readilyrecognized by a person of ordinary skill in the art.

[0050] The subject invention also pertains to a method for synthesizingcarboxymethylated aspartate chelating matrices, comprising the steps of:

[0051] (a) Michael addition of the α—amino function of monoprotectedα,ω—diamrino acids to maleic acid;

[0052] (b) deprotecting the ω—-amino functionality; and

[0053] (c) attaching the chelator primary amine molecule to a solidmatrix.

[0054] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

[0055] Example 1—Large-Scale Preparation of CM-Asp Chelating Resin

[0056] SEPHAROSE CL-6B or CL-6B (Pharmacia, 8.0 L) is washed with ddH20,suction dried, and transferred to a 22-L round bottom flask equippedwith mechanical stirring. Epichlorohydrin (about 2.0 L) is added, theSepharose resin mixed to a thick suspension, and allowed to stand atroom temperature for about 20 minutes. A solution of sodium hydroxide(about 560 g) and sodium borohydride (about 48 g) in approximately 6400mL ddH₂0 is added and the mixture stirred overnight at ambienttemperature. The oxirane-derivatized resin, collected by filtration, iswashed ten times with ddH,₂O (about 10 L each), once with 10% sodiumcarbonate (about 10 L), suction dried, and transferred to a 22L roundbottom flask. A specimen of the oxirane derivatized resin treated with1.3 M sodium thiosulfate is titrated to approximately pH 7.0 todetermine the oxirane concentration (preferably, >700 lmol/g).

[0057] To a solution of sodium hydroxide (approximately 268 g) in about7.6 L ddH₂0 is added L-aspartic acid (about 575 g) and sodium carbonate(about 1700 g), keeping the temperature below about 25° C. The pH isadjusted to approximately 11.0 and the solution added to the resin.Using mechanical stirring and a heating mantle, the reaction mixture isbrought to about 80° C. for 4 hours and allowed to cool to roomtemperature overnight. The resin was collected by filtration, washed tentimes with ddH₂O (about 10 L each), once with 10% sodium carbonate(about 10 L), suction dried, and transferred to a 22-L round bottomflask equipped with mechanical stiring.

[0058] To an ice-cooled solution of sodium hydroxide (about 900 g) in 12L ddH.O was added bromoacetic acid (about 3000 g) in approximately 750 gincrements, keeping temperature below about 30° C. Sodium carbonate(about 660 g) is added and the pH is adjusted to about 10. The resin isreacted with the solution at ambient temperature overnight. The resin iscollected by filtration, washed six times with ddH₂O (about 10 L each),six times with 10% acetic acid, and ten times with ddH₂0. Washing iscontinued with ddH₂O until the pH reached about 6.0 by litmus paper. TheCM-Asp chelating resin was suction dried in preparation for metalloading.

[0059] Example 2 —Preparation of C-Linked CM-Asp Chelating Resin

[0060] N⁶-Carbobenzyloxy-L-lysine (6.15 g) and excess maleic acid (17.6g) were dissolved in 2 M NaOH (35 mnL). The solution was refluxed for 24hr and allowed to cool to ambient. The pH was adjusted to 3 with 6 M HCland chilled. The mixture was filtered to remove the unreacted maleicacid which was washed with water (15 mL). The filtrate and washings werecombined and evaporated. The residue was dissolved in 4% ammoniumformate (120 iL) and degassed briefly. 10% Pd/C (600 mg) was added andthe mixture was refluxed under Ar with stirring for 5 hr. The mixturewas filtered through celite and the filtrate evaporated. The residue wasdissolved in a solution of sodium hydroxide (1.3 g) and sodium carbonate(8.7,,g) in dd H₂0 (40 mL). The final pH was adjusted to 1 1. Thissolution is added to the oxirane-derivatized resin (40 Ti bed volume)prepared as described in Example 1. The mixture was stirred at 60′ withmechanical stiirinfor 4hr and at ambient for16 hr. The resin wascollected by filtration, washed with ddH₂0 (6 x 1 00 nlL), IO% HOAc (6 xI100 mLi), and ddH₂0 (6 X I100 Ti) or until the pH reached about 6 bylitmnus paper test. The C- Linked CM-Asp chelating resin was suctiondried in preparation for metal loading.

[0061] Example 3 —Metal Loadingz of CM-AMp Chelatinga Resin

[0062] The CM-Asp chelating resin of Example 1 (about 1 L of suctiondried bed volume) is treated with a transition metal ion solution, eg.,2 L of either 200 mM of cobalt chloride hexahydrate, nickel sulfatehexahydrate, copper sulfate pentahydrate, or zinc chloride, according tothe metal ion deserved. The resin is reacted with the 200 mM metalsolution at room temperature for approximately 72 hours and thencollected by filtration. The metal loaded CM-Asp chelating resin iswashed five times with ddH₂O (about I L each), two times with 100 mMNaCl (about I L each), six times with ddH20 (about I L each), and oncewith 20% aq. ethanol (about I L). The resin can be stored in 20% aq.ethanol.

[0063] Example 4 —Comparison of Co²1 CM-Asp Resin With Ni²⁺ NTA onRecombinant 6XHis

[0064] Prgpro-a-Factor Under Denaturiniz Conditions Usintz pH GradientFor a qualitative comparison of the purification of Co+CM-Asp chelating,resin and Ni-NTA under denaturing conditions, the C-terminal,6XHis-tagged prepro-(Xfactor of S. cerevisiae was expressed in E. coli.One grain bacterial cell pellet was lysed in 6 M guanidinium-HCI(Gu-HCI) and 0. 1 M NaH{₂PO₄, pH 8.0. Three milliliters of clarifiedlysate was loaded onto a Co²⁺ CM-Asp chelating resin gravity flowcolumn. The resin-proteins mixture was washed with 8 M urea, 0. 1 MNaH₂PO₄, pH 8.0, and deluted with 8 M urea, 0.1I M NaH₂PO₄ at threedifferent pHs, 6.3, 5.9, and 4.5. Finally, all bound proteins weredeluted with 6 M Gu-HCI, 0.1I M NaH₂PO₄, 0.2 M acetic acid. Samples fromeach step were loaded on a 12% polyacrylamidelSDS gel, electrophoresed,and the gel was stained with Coomassie blue. The 6XHis-taggedprepro-a-factor was deluted at pH 6.3 as a single prominent band on thegel.

[0065] In the same manner, 3 ml of clarified lysate was loaded onto aNi-NTA gravity flow column. The resin-proteins mixture was washed anddeluted the same as above. Samples from each step were loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and the gel was stained withCoomassie blue. There were more than 10 protein bands in elution at pH6.3. The 6XHis-tagged prepro-cx-factor was a minor band among them. Themajority of the protein was deluted at pH 4.5 without any othercontaminant proteins. This demonstrated that the highly purified6Xis-tagged prepro-a-factor was deluted from Co²⁺ CM-Asp chelating resinat the conditions (pH 6.3) under which Ni-NTA was still releasingcontaminants. The affinity of Co²⁺ CM-Asp chelating resin to6XHis-tagged prepro-Ca-factor was more selective than Ni-NTA to theprotein.

[0066] Results show that highly purified 6is-tagged protein elutes fromCo²⁺ CM-Asp chelating resin while Ni-NTA is still releasingcontaminants. See FIGS. 2A-2B: FIG. 2A: Results after using 1 ml of Co²⁺CM-Asp chelating resin. FIG. 2B: Results after using 1 ml of nickel-NTA.

[0067] Example 5 —Recombinant 6XHis DHFR Purification with CM-Asp ResinUnder Native Conditions

[0068] N-terminal, 6XFis-tagged mouse dihydrofolate reductase (DHFR, MW20.3 kDa) was expressed in E. coli cells. Cells were then pretreatedwith 0.75 m]/ml lysozyme and disrupted in lysis buffer (100 mM NaH₂PO₄,10 mnM Tris-HCI, pH 8.0) by mechanical shearing, 800 jil of theclarified lysate was applied to 100 lil of Co²° CM-Asp chelating resin,pre-equilibrated with lysis buffer, and washed with one ml of lysisbuffer three times. All bound protein was deluted by 300 gl of 100 mMEDTA, pH 8.0. Twenty microliters of lysate and 40 pul of each subsequentfraction from elution were run on a 12% polyacrylamide/SDS gel. The gelwas stained with Coomassie blue. One single protein band was shown at aposition of MW 20.3 kDa. Results showed the selective binding affinityof Co²⁺ CM-Asp chelating resin to 6Xis-taaged DHFR under nativepurification conditions. No discemable binding of host proteinsoccurred.

[0069] Results show that Co²⁺ CM-Asp chelating resin has selectivebinding affinity for 6X Histidines. No discemable binding of hostproteins occurred. See FIG. 3.

[0070] Example 6—Recombinant 6XHis DIFR Purification with CM-Asp ResinUnder Denaturing Conditions

[0071] N-tenional 6XHis-tagged mouse DHFR was expressed in a 25-mnlculture ofE. coli. Cells were pelleted, resuspended in lysis buffer (100mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 8.0), and disrupted byvortexing. Six hundred microliters of clarified lysate were applied to aCo²⁺ CM-Asp chelating resin spin column containing 0.5 ml of Co²⁺CM-Asp;chelating resin-NX metal affinity resin and centrifuged for 2 minutes at2,000 x g. The column was washed twice with 1 ml of wash buffer (100 imMNaH₂PO₄, 10 nmM PIPES, pH 7.0), and bound proteins were deluted with 600Ill of elution buffer (20 miM PIPES, 100 nmM NaC1, 8 M urea, pH 6.0).Twenty microliters of lysate and 40 I1 of each subsequent fraction fromthe elution were loaded onto a 12% polyacrylaniide/SDS gel andelectrophoresed. The gel was stained with Coomassie blue. One singleprotein band was shown at the position of 20.3 kDa Results showed theselective binding affinity of Co²⁺ CM-Asp chelating resin to6XIis-tagged DHFR under denaturing conditions. The binding properties ofCo²CM-Asp chelating resin to 6X histidines allow proteins deluted undermild pH conditions (pH 6.0) that protect protein integrity.

[0072] Results show that bound protein can be deluted at mild pH (pH6.0). This indicates that the binding properties of Co²⁺ CM-Aspchelating resin allow protein elution under mild pH conditions thatprotect protein integrity. See FIG. 4.

[0073] Example 7—Recombinant 6XHis DHFR Purification with CM-Asp ResinUnder Native Conditions with Increasing Concentrations ofBeta-Mercaptoethanol N-terminal, 6XHis-tagged mouse DHFR was expressedin E. coli. Twenty-five milliliters of cell culture were disrupted in 2ml of sonication buffer (100 MM NaH₂PO₄, 1 0 mM Tris-HCl, and 100 miMNaCl, pH 8.0) by freezing and thawing. Then, 2.66 ml of clarified lysatewere applied to a 200-el Co²⁺ CM-Asp chelating resin gravity flowcolumn, pre-equilibrated with the sonication buffer. The proteins/resinmixtures were washed three times with sonication buffer, pH 8.0. Allbound proteins were deluted with 600 pI of 100 mM EDTA, pH 8.0. To testthe effect of P-mercaptoethanol on the Co²° CM-Asp chelating resinpurification under native conditions, all buffers used here containedeither 0, 10, 20, or 30 mM P-mercaptoethanol. Samples from each elutionwere electrophoresed on a 12% polyacrylamidelSDS gel, and the gel wasstained with Coomassie blue. One single protein band at the position ofMW 20.3 kDa was shown from all elutions. The presence of P-mercaptoethanol did not obsolete the purity of 6XHis-tagged DHFRpurified by Co²⁺ CM- Asp chelating resin. With up to 30 rnMP-mercaptoethanol in all purification buffers, there was no predominantband at 20.3 kDa in flowthroughs, indicating that no loss of metaloccurred during protein purification using Co²⁺ CM-Asp chelating resinin the presence of I-mercaptoethanol.

[0074] Results show that with up to 30 mM ,-mercaptoethanol in thepurification buffer, there is no predominant band at 20.3 kDa in theflowthrough, indicating that no loss of metal occurred during proteinpurification using Co²⁺ CM-Asp chelating resin in the presence ofP-mercaptoethanol. See FIG. 5.

[0075] Example 8 —Yields of 6XHis DHFR From Cell Lvsates Purified byCM-Asp Versus Ni- NTA in the Presence of Beta-Mercaptoethanol

[0076] N-terminal, 6XHis-tagged DHFR was expressed and purified by Co²⁺CM-Asp chelating resin under native conditions as described in Example7. Protein concentrations were determined by Bradford assay. Yields wereexpressed as a percentage of total protein in the cell lysate. Theyields of purified 6XHis-tagged DBFR were 14%, 28%, 34%, and- 35%respectively, with P-mercaptoethanol present in purification buffers atthe concentrations of 0, 10, 20, and 30 mM. The protein was purified byNi-NTA under the same native conditions; the yields of purified6XHis-tagged DHFR were 4%, 8.8%, 3.4%, and 4% respectively withP-mercaptoethanol present in purification buffers at the concentrationsof 0, 10, 20, and 30 mM. The yields of purified ⁶XHis-tagged DEFR weresignificantly higher when using C02+CM-Asp chelating resin compared toNi-NTA under native purification conditions with P-mercaptoethanol. Thisindicates that the metal ion on Co²⁺ CM-Asp chelating resin is stronglyanchored to SEPHAROSE beads by a CMI-ASP metal chelator that is idealfor binding octahedral metals.

[0077] Results show that the yields of purified 6XHis DHFR aresignificantly higher at 10, 20, and 30 mM β-mercaptoethanol usingC02+CM-Asp chelating resin compared to using Ni-NTA. This indicates thatthe metal ion on Co²⁺ CM-Asp chelating resin is strongly anchored tosepharose beads by CM-Asp metal chelator that is advantageous forbinding octahedral metals. See FIG. 6.

[0078] Example 9 —Purification of 6XHis GFP bv Co²-CM-Asp Under NativeConditions N-terminal, 6XHis-tagged green fluorescent protein (GFP) wasexpressed in E. coli cells. Cells were pelleted, resuspended insonication buffer (100 mM NaH2PO4, 10 rnM Tris-HCI, and 100 mM NaCl, pH8.0), and disrupted by freezing and thawing three times. Two millilitersof clarified lysate were applied to 400 pl of Co²⁺ CM-Asp chelatingresin, pre-equilibrated with sonication buffer, and washed three timeswith 2 ml of sonication buffer, pH 8.0. The 6XHis-tagged GFP was delutedwith 400 gl of 75 niM imidazole buffer containing 20 mM Tris-HCl and 100mM NaCl, pH 8.0. Samples from each purification step were loaded onto a12% polyacrylamide/SDS gel, electrophoresed, and the gel was stainedwith Coomassie blue. One single band was shown at the position of MW27.8 kDa in the elution with 75 miM imidazole. This demonstrated that6XMis-tagged GFP selectively bound on Co²⁺ CM-Asp chelating resin andcan be deluted with low concentration of imidazole under nativepurification conditions.

[0079] Samples from each purification step were also loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and transblotted to a PVDFmembrane. The proteins on the blot were probed with anti-GFP monoclonalantibody. One single GFP band was clearly shown in the samples of celllysate and elution. There was no GFP band shown in flowthrough, whichindicated that all expressed GFP in cell lysate was bound to Co²⁺ CM-Aspchelating resin.

[0080] Results show that (FIG. 7A) Coomassie blue stained gel shows onesingle band in 75 riuM imidazole elution. This indicates that 6XHistidines selectively bound on Co²⁺ CM- Asp chelating resin. Westernanalysis data shows no GFP in flowthrough which indicates the highaffnity between Co²⁺ CM-Asp chelating resin and 6X histidines (FIG. 7B).

[0081] Example 10 —Biological Activity of 6XHis GFP Purified bv Co²⁺CM-Asp

[0082] N-terminal, 6XHis-tagged GFP was expressed in E. coli. Celllysate was prepared as described in Example 7. The cell lysate wasloaded onto a 2-ml Co²⁺ CM-Asp chelating resin Disposable GravityColumn, and purified using the Batch/Gravity Flow column purificationmethod as described in Example 9. The column was washed with sonicationbuffer three times and deluted with 100 mM EDTA, pH 8.0. Samples werecollected in microfuge tubes from each purification step. Thefluorescence of all collected samples was detected using an UltraLumElectronic U.V. Transilluminator. Samples of cell lysate and elutionshowed strong fluorescence. This experinent demonstrated that6XHis-tagged GFP can be purified to homogeneity by Co²′ CM-Asp chelatingresin under native conditions and maintains biological activity.

[0083] The photo of samples from each purification step shows that GFPcan be purified to homogeneity by Co²° CM-Asp chelating resin undernative conditions, and the fluorescence indicates that GFP purified byCo²′ CM-Asp chelating resin still maintains its biological activity. SeeFIG. 8.

[0084] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

1. An immobilized metal ion affity chromatography purification methodfor purification of a recombinant proteins, said method comprising: (a)providing carboxymethylated aspartate ligand complexed with a transitonmetal ion in a ²⁺ oxidation state, having a coordination number of 6;(b) loading a mixture of cell lysate comprising a recombinant proteinhaving a polyhistidine tail to bind with said ligand; and (c) elutingsaid recombinant protein with a suitable elutant to obtain a purifiedrecombinant protein.
 2. The method, according to claim 1, wherein saidtransition metal-complexed carboxymethylated aspartate ligand forms acarboxymethylated aspartate chelating matrix which comprises saidtransition metal and a polymer matrix.
 3. The method, according to claim2, wherein said transition metal is connected to said polymer matrix bya linking arm and a finctional inking group.
 4. The method, according toclaim 3, wherein said linking arm is selected from the group consistingof-CH₂CH(OH)CH₂-, -CH₂(OH)CH₂-0-CH2CH(OH)CH2-, CH₂)₄NHCH₂CH(OH)CH₂-, andCH₂)₂NHCH₂CH(OH)CH₂-.
 5. The method, according to claim 3, wherein saidfinctional linking group is selected from the group consisting of 0, S,and NH.
 6. The method, according to claim 2, wherein said polymer matrixis agarose.
 7. The method, according to claim 2, wherein saidcarboxymethylated aspartate chelating matrix has the structure

wherein: R₄-R₅-R6 =H M=transition metal ion in a 2+ oxidation state witha coordination number of 6; R₁=a linking arm connecting the nitrogenatom of CM-Asp with R₂; R₂=a functional inking group tbrough whichCM-Asp linng arm RI is connected to R₃; and R₃=a polymer matrix
 8. Themethod, according to claim 2, wherein said carboxymethylated aspartatechelating matrix has the structure

wherein: R₁−R₂-R₃ =H; M=transition metal ion in a 2+ oxidation statewith a coordination number of 6; R4=a linldng arm connecting themethylene carbon atom of the carboxymethyl group of CM-Asp with R₅; R₅=afunctional linking group through which CM-Asp linking arm R4 isconnected to &; and R₆=a polymer matrix.
 9. An immobilized metal ionaffinity chromatography complex comprising a carboxymethylated aspartateligand and a tnansition metal complexed thereto, wherein said transitionmetal ion has a 2+ oxidation state and a coordination number of
 6. 10.The complex, according to claim 9, wherein said complex has thestructure:

wherein: R4-Rs-R6 H M=transition metal ion in a 2+ oxidation state witha coordination number of 6; R₁a linking arm connecting the nitrogen atomof CM-Asp with R₂; R₂=a fnctional linking group through which CM-Asplinking arm RI is connected to R₃; and R₃=a polymer matrix
 11. Themethod, according to claim 1 0, wherein said polymer matrix comprises apolymer matrix suitable for use in afinty or gel chromatography.
 12. Thecomplex, according to claim 10, wherein M=Fe²⁺ , Col+, Ni²⁺ , Cue, orZne; R=CH₂CH(OH)CH₂-, H₂(OH)CH₂ H₂CH(OH)CH2-, or CH₂)₂NHCH₂CH(OH)CH,-R₂=O, S, or NH; and R₃=agarose or polystyrene.
 13. The complex,according to claim 12, wherein M=Co²⁺ ; R₁=CH₂CH(OH)CH₂; R₂=O; andR₃=agarose, cross-linked or polystyrene
 14. A method for synthesizingcarboxymethylated aspartate agarose chelating resin, said methodcomprising (a) forming oxirane-agarose; (b) conjugating aspartic acid tooxirane-agarose; and (c) washing said aspartic acid-oxirane-agaroseconjugate to remove extraneously bound metals using a high ionicstrength solution.
 15. The method, according to claim 14, wherein saidconditions for oxirane-agarose formation comprise carrying out theformation at about room temperature, overnight, adjusting to about pH7.0.
 16. The method, according to claim 14, wherein said temperaturecontrol conditions for conjugating aspartic acid to said oxirane-agarosecomprise mixing at less than about 25° C., reacting at about 80° C. for4 hours, then cooling to room temperature overnight.
 17. The method,according to claim 14, wherein said washing step (c) comprises use of asolution of at least 7.5% sodium hydroxide.
 18. The complex according toclaim 9, wherein said complex has the structure:

wherein: R₁-R₂-R₃ =H; M=transition metal ion in a 2+ oxidation statewith a coordination number of 6; R₄=a linking arm connecting themethylene carbon atom of the carboxymethyl group of CM-Asp with R₅; R₅=afinctional linking group through which CM-Asp linking arm R4 isconnected to R6; and 6=a polymer matrix.
 19. The method, according toclaim 18, wherein said polymer matrix comprises a polymer matrixsuitable for use in affinity or gel chromatography.
 20. The complexaccording to claim 18, wherein M=Fe²⁺ , Co²⁺ , Ni²⁺ , Cu²⁺ , or Zn²⁺ ;R₄=dCH2)₄NHCH2CH(OH)CH₂- or dCH₂)₄NH-; R₅=O, S, NH, or CO; andR6=agarose or polystyrene.
 21. The complex, according to claim 20,wherein M=Co²⁺ ; R={CH₂)₄NHCH₂CH(OH)CH₂- or {CH₂)₄NH-; R₅=O or CO; andR6=agarose, cross linked, or polystyrene.
 22. A method for synthesizingcarboxymethylated aspartate chelating matrices, said method comprisingthe steps: (a) Michael addition of the a-amino function of monoprotecteda,u-diamino acids to maleic acid; (b) deprotecting the w-aminofimctionality; and (c) attaching the chelator primary amine molecule toa solid matrix.
 23. A method for screening for protein finction on amicrotiter plate or filter, said method comprising the steps: (a)immobilizing a complex of claim 1 to the plate or filter; (b) bindingsaid immobilized complex to the protein for which the function is beingscreened; and (c) performing an assay for protein finction on the boundprotein. HAtSHAPPS ci5cl.doc/DNB/clr